Cereblon
The primary molecular target of Thalidomide-5-OH is cereblon (CRBN), an E3 ubiquitin ligase substrate receptor. As a ligand for the E3 ubiquitin ligase complex, this compound binds to CRBN and recruits the ubiquitin-proteasome system. In the context of PROTAC technology, the thalidomide moiety serves as the E3 ligase recognition element, while the hydroxyl group provides a handle for conjugation to a linker and subsequently to a target protein ligand. This design enables the targeted degradation of specific proteins by bringing the target protein into proximity with the E3 ligase.

Angiogenesis, in particular anti-angiogenesis, is an area of particular therapeutic interest in cancer treatment. Several anti-angiogenic agents are in the final stages of clinical trials. One of these agents, thalidomide, best known for its teratogenic potential, is showing promise against several tumor types. Thalidomide has been shown previously to require bio-activation to exert its anti-angiogenic effect in isolated blood vessels and endothelial cells. In this work, we confirmed these findings using the in utero chicken embryo chorioallantoic membrane (CAM) system. In particular, the anti-angiogenic effect of thalidomide is significantly enhanced by activation by human but not by rat liver microsomes. We also showed in the CAM assay that hydroxylation of thalidomide at either the 1'- or 5-position retained anti-angiogenic activity whereas its hydroxylation at the 4-position led to an inactive compound. We further demonstrated that thalidomide shows weak anti-proliferative activity against MDA-MB-231 human breast cancer cells in culture. Thalidomide showed slightly more anti-proliferative activity, however, against the SH-SY5Y human neuroblastoma and human umbilical vein endothelial cell (HUVEC) types. Furthermore, incubation of thalidomide with human liver microsomes added no additional anti-proliferative effect in these cell types versus thalidomide given alone. Finally, we report that none of the thalidomide metabolites tested had any anti-proliferative effect against the breast or neuroblastoma cells, but do possess appreciable anti-proliferative activity against the endothelial cells. In summary, this work suggests that hydroxylated thalidomide analogs based on putative metabolites of the drug possess significant anti-angiogenic activity and that exploring further derivatives of these as potential anti-angiogenic agents warrants further merit[1].

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In vitro studies demonstrate that Thalidomide-5-OH functions as a cereblon ligand in PROTAC molecules. The compound binds to the E3 ubiquitin ligase via the CRBN protein. The hydroxyl group at the 5-position allows for conjugation to various linkers, which can then be connected to target protein ligands. This compound is designed to facilitate targeted protein degradation in cellular systems through the ubiquitin-proteasome pathway. It is typically used as a synthetic intermediate or building block in the preparation of complete PROTAC molecules.

The teratogenicity of the chemotherapeutic drug thalidomide is species-specific and affects humans, non-human primates, and rabbits. The primary oxidation of thalidomide in previously investigated rodents predominantly resulted in the formation of deactivated 5'-hydroxythalidomide. In the current study, similar in vivo biotransformations to 5-hydroxythalidomide and 5'-hydroxythalidomide were confirmed by the analysis of blood plasma from male rabbits, a thalidomide-sensitive species, after oral administration of thalidomide (2.0 mg/kg). Similar levels of thalidomide in seminal plasma and in blood plasma were detected using liquid chromatography-tandem mass spectrometry at 4 hr and 7 hr after oral doses in male rabbits. Seminal plasma concentrations of 5-hydroxythalidomide and 5'-hydroxythalidomide were also seen in male rabbits in a roughly similar time-dependent manner to those in the blood plasma after oral doses of thalidomide (2.0 mg/kg). Furthermore, the values generated by a simplified physiologically based pharmacokinetic rabbit model were in agreement with the measured in vivo blood plasma data under metabolic ratios of 0.01 for the hepatic intrinsic clearance of thalidomide to both unconjugated 5-hydroxythalidomide and 5'-hydroxythalidomide. These results suggest that metabolic activation of thalidomide may be dependent on rabbit liver enzymes just it was for cytochrome P450 enzymes in humanized-liver mice; in contrast, rodent livers predominantly mediate biotransformation of thalidomide to 5'-hydroxythalidomide. A developmental toxicity test system with experimental animals that involves intravaginal exposures to the chemotherapeutic drug thalidomide via semen should be considered in the future[2].

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In vivo activity data for Thalidomide-5-OH as a standalone compound are not reported, as it is utilized as a synthetic intermediate or ligand building block in PROTAC design rather than as a therapeutic agent itself. The in vivo efficacy of PROTAC molecules incorporating this thalidomide-based ligand would depend on the specific target protein ligand and the overall pharmacokinetic properties of the complete PROTAC construct. As a ligand building block, its primary role is to facilitate targeted protein degradation through E3 ligase recruitment.

Thalidomide causes teratogenic effects by inducing protein degradation via cereblon (CRBN)‐containing ubiquitin ligase and modification of its substrate specificity. Human P450 cytochromes convert thalidomide into two monohydroxylated metabolites that are considered to contribute to thalidomide effects, through mechanisms that remain unclear. Here, we report that promyelocytic leukaemia zinc finger (PLZF)/ZBTB16 is a CRBN target protein whose degradation is involved in thalidomide‐ and 5‐hydroxythalidomide‐induced teratogenicity. Using a human transcription factor protein array produced in a wheat cell‐free protein synthesis system, PLZF was identified as a thalidomide‐dependent CRBN substrate. PLZF is degraded by the ubiquitin ligase CRL4CRBN in complex with thalidomide, its derivatives or 5‐hydroxythalidomide in a manner dependent on the conserved first and third zinc finger domains of PLZF. Surprisingly, thalidomide and 5‐hydroxythalidomide confer distinctly different substrate specificities to mouse and chicken CRBN, and both compounds cause teratogenic phenotypes in chicken embryos. Consistently, knockdown of Plzf induces short bone formation in chicken limbs. Most importantly, degradation of PLZF protein, but not of the known thalidomide‐dependent CRBN substrate SALL4, was induced by thalidomide or 5‐hydroxythalidomide treatment in chicken embryos. Furthermore, PLZF overexpression partially rescued the thalidomide‐induced phenotypes. Our findings implicate PLZF as an important thalidomide‐induced CRBN neosubstrate involved in thalidomide teratogenicity[3].

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In vitro assays for evaluating Thalidomide-5-OH typically involve binding studies to assess its interaction with cereblon (CRBN), the E3 ubiquitin ligase substrate receptor. Surface plasmon resonance (SPR) or fluorescence polarization techniques can be used to measure the binding affinity (Kd) between the thalidomide-based ligand and CRBN. Competition binding assays using fluorescently labeled probes are also commonly used to determine the inhibitory concentration (IC50) of the ligand for CRBN binding. The hydroxyl group can be used for conjugation studies to evaluate linker attachment efficiency.

To examine the 5‐hydroxythalidomide‐dependent degradation of overexpressed PLZF, SALL4 or IKZF1, HEK293T‐CRBN−/− cells were cultured in 48‐well plates and transfected with 200 ng pcDNA3.1(+)‐FLAG‐CRBN‐WT and 20 ng pcDNA3.1(+)‐AGIA‐SALL4, pcDNA3.1(+)‐AGIA‐PLZF or pcDNA3.1(+)‐AGIA‐IKZF1. After the cells were transfected for 8 h, they were treated with thalidomide, 5‐hydroxythalidomide or DMSO (0.1%) in the culture medium at the indicated times and concentrations (Fig 4C). For endogenous SALL4, PLZF, IKZF1, HuH7 or THP‐1 cells were cultured in 48‐well plates and treated with thalidomide, 5‐hydroxythalidomide or DMSO (0.1%) in the culture medium at the indicated times and concentrations (Fig 4D and E).[3]

To examine the species specificity of IMiD‐dependent protein degradation, HEK293T‐CRBN−/− cells were cultured in 48‐well plates and transfected with 200 ng pcDNA3.1(+)‐FLAG‐(mouse or chicken) CRBN‐WT or CRBN‐IV and 20 ng pcDNA3.1(+)‐AGIA‐(mouse or chicken) PLZF or pcDNA3.1(+)‐AGIA‐(mouse or chicken) SALL4. After the cells were transfected for 8 h, they were treated with thalidomide or DMSO (0.1%) in the culture medium for the times and concentrations indicated in Fig 5A–D.[3]

To examine whether 5‐hydroxythalidomide induced the degradation of mouse or chicken SALL4 or PLZF, HEK293T‐CRBN−/− cells were cultured in 48‐well plates and transfected with 200 ng pcDNA3.1(+)‐FLAG‐(mouse or chicken) CRBN‐WT and 20 ng pcDNA3.1(+)‐AGIA‐(mouse or chicken) SALL4 or pcDNA3.1(+)‐AGIA‐(mouse or chicken) PLZF. After the cells were transfected for 8 h, they were treated with thalidomide, 5‐hydroxythalidomide or DMSO (0.1%) in the culture medium for the times and concentrations indicated in Fig 5E–H.[3]

In all experiments, cells were lysed by boiling in 1× sample buffer containing 5% 2‐mercaptoethanol and the lysates were analysed by immunoblotting.

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Quantitative real‐time PCR (qRT–PCR)[3]
To demonstrate decreased post‐translational level of PLZF protein, PLZF mRNA expression in HEK293T, HuH7 or THP‐1 cells treated with DMSO or lenalidomide for 24 h was assessed by qRT–PCR. To analyse SALL4 or PLZF mRNA expression, HuH7 cells treated with DMSO, thalidomide or 5‐hydroxythalidomide for 24 h were assessed by qRT–PCR. Total RNA was isolated from HEK293T, HuH7 or THP‐1 cells treated with DMSO or lenalidomide for 24 h using a SuperPrep cell lysis kit. cDNA was synthesised using a SuperPrep RT kit according to the manufacturer's protocol. RT–PCR was performed using a KOD SYBR qPCR Mix, and data were normalised against glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) mRNA levels. PCR primers were as follows: PLZF FW 5′‐GCACAGTTTTCGAAGGAGGA‐3′, PLZF RV 5′‐GGCCATGTCAGTGCCAGT‐3′, SALL4 FW 5′‐GGTCCTCGAGCAGATCTTGT‐3′, SALL4 RV 5′‐GGCATCCAGAGACAGACCTT‐3′, GAPDH FW 5′‐AGCAACAGGGTGGTGGAC‐3′, GAPDH RV 5′‐GTGTGGTGGGGGACTGAG‐3′.

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In vitro cell-based assays for Thalidomide-5-OH typically involve its incorporation into PROTAC molecules followed by evaluation of target protein degradation in cultured cells. Cells are treated with PROTACs containing this thalidomide-based ligand, and the levels of the target protein are measured by Western blotting or immunofluorescence to assess degradation efficiency. Dose-response experiments are performed to determine the DC50 (half-maximal degradation concentration) of the PROTAC construct. Additionally, cell viability and proliferation assays may be conducted to evaluate the functional consequences of target protein degradation.

Thalidomide treatment of chicken embryos[3]
A solid crystal of thalidomide was resolved in 45% 2‐hydroxypropyl‐beta‐cyclodextrin for 1–2 h at 60°C to make thalidomide (2 μg/μl, 7.8 mM) or 5‐hydroxythalidomide (2 μg/μl, 7.3 mM) stock solution. This stock was mixed with the same volume of 2× Hanks buffer as a working solution (1 μg/μl, 3.9 mM thalidomide or 3.6 mM 5‐hydroxythalidomide). To apply thalidomide to an embryo, a small hole was opened at the amnion above the right forelimb bud at HH st. 18 (E3), and the working solution was injected in a space between the amnion and right forelimb bud. In the case of samples for in situ hybridisation, to induce a strong effect on target proteins of thalidomide or 5‐hydroxythalidomide for short exposure time, immunofluorescence and immunoblot analyses, embryos were treated with 30 μl of working solution and incubated until HH st. 22/23 (E4). In the case of samples of skeletal pattern analysis, to reduce lethality and increase the collection rate of chicken embryos showing teratogenic phenotypes in limb bud at HH st. 36 (E10), embryos were treated three times with 10 μl of working solution every 12 h and incubated until approximately HH st. 36 (E10).

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In vivo animal studies using Thalidomide-5-OH are conducted as part of the evaluation of complete PROTAC molecules that incorporate this thalidomide-based ligand. Typical protocols involve administering PROTAC constructs to mouse xenograft models or disease-relevant animal models, followed by assessment of target protein degradation in harvested tissues via Western blot or immunohistochemistry. Pharmacodynamic endpoints include measurement of target protein levels, downstream signaling pathway modulation, and tumor growth inhibition in efficacy studies. Dosing regimens are optimized based on the pharmacokinetic properties of the specific PROTAC construct.

As a ligand building block rather than a therapeutic drug, comprehensive pharmacokinetic data for Thalidomide-5-OH alone are limited. The compound has a molecular formula of C13H10N2O5 and a molecular weight of 274.23. When incorporated into PROTAC molecules, the thalidomide-based ligand contributes to the overall physicochemical properties of the complete construct. The hydroxyl group provides a handle for linker conjugation, which can influence the solubility and pharmacokinetic properties of the complete PROTAC molecule.

The toxicity profile of Thalidomide-5-OH as an individual compound is not extensively characterized, as it is primarily used as a research reagent and synthetic building block. The compound is intended for research use only and is not approved for therapeutic use in humans. The thalidomide core structure is known to have teratogenic effects, and appropriate safety precautions should be taken when handling this compound. Standard laboratory safety practices, including the use of personal protective equipment and handling in a fume hood, are recommended.

[1]. Effects of Putative Hydroxylated Thalidomide Metabolites on Blood Vessel Density in the Chorioallantoic Membrane (CAM) Assay and on Tumor and Endothelial Cell Proliferation. Biol Pharm Bull. 2002 May;25(5):597-604.

5-Hydroxythalidomide is a type of phthalimide compound.

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Thalidomide-5-OH (CAS 64567-60-8) has a molecular formula of C13H10N2O5 and a molecular weight of 274.23. The compound has a purity of >95%. It is a thalidomide-based cereblon ligand used in the recruitment of CRBN protein and can be connected to target protein ligands through a linker to form PROTACs. The compound is part of the E3 ligase ligand building block category and is used in PROTAC synthesis for targeted protein degradation research.

C13H10N2O5

274.2289

274.059

C, 56.94; H, 3.68; N, 10.22; O, 29.17

64567-60-8

Thalidomide-4-OH;5054-59-1

5743568

Gray to brown solid powder

1.611g/cm3

600ºC at 760mmHg

316.7ºC

1.679

0.007

2

5

1

20

503

0

O=C1CCC(N2C(=O)C3C=CC(=CC=3C2=O)O)C(=O)N1

LJBQRRQTZUJWRC-UHFFFAOYSA-N

InChI=1S/C13H10N2O5/c16-6-1-2-7-8(5-6)13(20)15(12(7)19)9-3-4-10(17)14-11(9)18/h1-2,5,9,16H,3-4H2,(H,14,17,18)

2-(2,6-dioxopiperidin-3-yl)-5-hydroxyisoindole-1,3-dione

5-hydroxythalidomide; 64567-60-8; 2-(2,6-dioxopiperidin-3-yl)-5-hydroxyisoindoline-1,3-dione; Thalidomide-5-OH; 2-(2,6-dioxopiperidin-3-yl)-5-hydroxyisoindole-1,3-dione; (+/-)-Hydroxythalidomide; CHEMBL182442; 29V976C3CJ;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :~62.5 mg/mL (~227.91 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.58 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.58 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)